Unitary one-piece sample mini-column

ABSTRACT

This invention is directed to a unitary one-piece sample mini-column for a cassette chemical immobilization and treatment system for enabling the performance of various complex chemistries. The cassette assembly comprises a plurality of analyte sample columns (&#34;mini-columns&#34;), reagent wells containing pre-packaged reagents including powdered, microencapsulated, liquid or lyophillized reagents, analyte loading funnels, alignment mechanisms for the analyte sample columns, and a machine readable instruction code set for identfying a chemical treatment protocol. The mini-columns are improved columns having a high pressure interface capability to permit direct insertion of the mini-column into a high-pressure solvent line for use as a support column for HPLC analysis. The same chemistries may be performed at all sample mini-column addresses, or a separate, completely independent set of protocols may be defined for each address, or for each block of addresses pursuant to the instructions contained in the machine readable code on the cassette.

This is a division, of application Ser. No. 08/679,355 filed on Jul. 9,1996, and now U.S. Pat. No. 5,800,784.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a cassette-type chemical sampletreatment system apparatus and method for use in the treatment of lessthan one milligram quantities of amino acids, proteins, peptides, andthe like pursuant to predetermined or preselected chemical, biochemical,and biomedical protocols. More particularly, the invention of thisapplication relates to a cassette-type chemical sample treatment systemand method having a cassette for holding a plurality of sample columnsfor immobilizing preselected samples and a plurality of reagent wellsfor retaining preselected reagents for enabling predeterminedchemistries of the preselected samples with the preselected reagents,including column loading, treatment, and post-treatment analysis of thereacted sample with near-zero dead volume and minimal humanintervention, the predetermined chemistries being specified by machinereadable code integrated into the cassette.

2. Background Art

With the exception of certain high-performance liquid chromatography(HPLC), proteins and peptides are typically fractionated in aqueousbuffers containing amines, salts and, often, denaturants. Therefore,additional manipulations such as desalting by HPLC, precipitation, ordialysis are required to render the sample matrix compatible withprotein and peptide chemistry or peptide sequencing protocol. Each ofthese additional steps, however, involves potential losses, especiallywhen only less than milligram amounts of protein are present at lowconcentrations. It is generally understood that proteins includepeptides, accordingly, for purposes of this invention, no distinction ismade between peptides and proteins, and reference to one will also applyto the other.

Many of the steps involved with the aforementioned steps have beeneliminated by immobilizing a protein or a peptide directly onto a solidsupport, washing away any interfering components, and leaving theprotein bound to the support ready for either further on-columnchemistries or removed for analysis. Hewlett-Packard AnalyticalInstruments (Palo Alto, Calif., U.S.) manufactures a family ofanalytical devices (HP-G1000A, HP-G1004B, and HP-G1005A) which usesample columns containing a solid support upon which proteins orpeptides might be immobilized. The HP-G1004A is a Protein ChemistryStation (PCS) which permits on-column chemistries to proteins andpeptides immobilized on a hydrophobic support. The HP-G1005 performsstandard Edman degradation protocols on a peptide immobilized on abi-phasic support for the performance of peptide sequencing.

Post-translational modifications will determine the stereochemistry andconformation of the peptide. Accordingly, there is a need to determinethe nature of the side groups so that a conformational analysis or astructure determination may be made. Such post-translationalmodification chemistries may be carried out manually orsemi-automatically whereby a sample is subjected to reaction with theappropriate reagents to give an appropriate indication in the event thepost-translational modification is present. The HP-G004 ProteinChemistry Station (PCS) represents the state of the art with respect toenabling semi-automatic performance of chemistries. The PCS haslimitations, however, with respect to the types and complexity ofchemistries that might be performed on this system. For example, the PCSis not able to perform bidirectional pumping; it can only pump down.This limitation precludes shuttling sample or reagents back and forththrough the support. This feature would be desirable, for example, whenthe reactivity of a side group is affected by the polarity of a solventand the appropriate solvent is one that may separate the sample from thesupport. Second, the chemistries performed in the PCS require that thereagents be dispensed into a large funnel in order to be introduced tothe reaction. Without changing-out the funnel between reagents, asignificant risk of cross-contamination is created.

The HP PCS system employs a hydrophobic column to immobilize the sampleduring loading and during application of the chemistry. FIG. 1 is across-sectional diagram of single sample reaction chamber typical of thebackground art. A funnel a is press-fit attached to the inlet side ofthe hydrophobic column b, with the throat of the funnel c incommunication with the top opening of the column d. The funnel/columnassembly is loaded into a center cavity e of a clear Lucite reactionchamber f, the assembly being secured by compressing and twisting thefunnel/column assembly in a single movement into a bayonet-typeconnection on the walls of the Lucite holder g, and the column beingurged against the funnel by a spring k. A cap h is screwed onto thereaction chamber so as to seal the system. Inlet ports i,j in the cappermit the introduction of sample into the funnel and the introductionof a pressurized inert gas to pump the reagent through the column.During the chemistries performed on the PCS, the funnel is neverchanged-out. As a result, a serious concern with the system and methodof the background art is that the side walls of the funnel may retainresidues of previously introduced reagents, thus resulting incross-contamination or reagents.

After insertion and bayonet-locking of the funnel/column assembly intothe reaction chamber, a protein or peptide solution sample is loaded inthe 5 ml funnel attached to the column. The cap is screwed onto theholder and over the funnel so that pressurized nitrogen, or other inertgas, may be applied to the sample and pressure forced into and throughthe hydrophobic sample column. The sample loading process capturesproteins and peptides on the hydrophobic portion of the sample column,while the sample solvent passes through.

It is possible to do multiple sample additions for larger volumes or touse a second solution to wash the sample. First, the holder cap removed,and a second sample or aqueous wash is added to the funnel, the capreattached to the holder, and the holder pressurized with nitrogen, thusforcing the aqueous wash through the hydrophobic sample column.

Following the sample loading, sample preparation (rinses), and possiblesample pre-treatment, the funnel/column assembly is removed from thesample reaction chamber and is transferred to another reaction chamberin the Protein Chemistry Station, wherein the appropriate reagents areadministered to perform the desired chemistries. A technician selects acomputer program which directs the PCS, via a micro-controllerinterface, to dispense the appropriate reagent into the sample funnelpursuant to the selected program. The appropriate reagent is directedthrough a tube and into a reagent port in the cap of the reactionchamber. The reagent flows from the reagent port into the sample funnel.Pressurized inert gas then forces the reagent out of the sample funneland into the column.

All chemistries are carried out in the column or the funnel, and withinthe reaction chamber. Also, since only one reaction chamber may beloaded at a time in the PCS, preparation and treatment of multiplesample columns is a time consuming and tedious effort. Further, the PCSprovides only four reservoirs for reagents, buffers or solvents, all ofwhich must be a liquid. Additionally, there may be reactions, especiallyof biomedical interest, wherein a solid reagent, such as a lyophilizedenzyme, vaccine, hormone preparations, and the like which exhibit lowerstability in solution, tending to either degrade rapidly or require lowstorage temperatures in its hydrated state, may be needed in order toexecute the desired chemistry. The devices of the background art are notable to accommodate these dry reagents. Accordingly, there is a need foran automated chemical treatment system capable of performing amultiplicity of both peptide and post-translational modificationchemistries sequentially on a plurality of samples, in an uninterruptedmanner with minimal human intervention or direction. The automatedchemical treatment system would also provide means to performchemistries external to the sample columns and for reintroduction of thereacted sample or analyte to the sample column. Also, there is a needfor an automated system that would provide means for "just-in-time"delivery of reagents requiring just-in-time solubilization, such asadsorbed, lyophilized or other powdered reactants to the sample.

Since the same sample funnel is used for all reagents there is a risk ofcross-contamination that may affect the outcome of sensitivechemistries. Minute quantities of reagent may adhere to the walls of thefunnel, only to be eluted into the sample column when the next reagentis introduced into the funnel. Where less than one milligram quantitiesof peptides or proteins are being investigated, the presence of minuteamounts of impurities or cross-contaminants may have a significantimpact on the results. Accordingly, there is a need for a samplecolumn/reaction chamber system that permits near-zero dead volume tominimize risk of cross contamination and the resulting inaccuracies suchcontamination may cause.

Once the protein chemistries are complete, the sample column from thePCS is removed from the sample reaction chamber and is transferred tothe appropriate analytical measurement device in order to measure orcharacterize the results of the chemistries performed. Typically, theanalysis is selected to characterize the products of a peptide cleavage.

Typically, RP-HPLC is used to analyze the reaction products. Ideally, itwould be desirable to insert the sample column of background artdirectly into an HPLC sample column holder, thus integrating the samplecolumn into the HPLC and transforming the sample column into a RP-HPLCcolumn. In order to obtain adequate separation of proteins, however,column pressures greater than 1500 psi are required. The sample columnof the background art rated to withstand pressures up to approximately1000 psi. At pressures greater than 1000 psi, the non-tapered end of thesample column typically fails. In order to operate at the extremely highpressures required for protein separation, a special adapter isrequired, and is attached to the inlet end of the sample column toaccommodate a high pressure line fitting. FIG. 2 shows a cross-sectiondiagram of the sample column and adapter typical of the background art.The adapter 1 is inserted into the non-tapered end d of the samplecolumn b. Since the sample column is a pre-column to the chromatographycolumn of the HPLC, the adapter is also a part of the HPLC pre-column.Accordingly, the column plus adapter of the background art is suppliedin addition to the standerd chromatogragh column rather than as asubstitute column. Consequently, the adapter 1 must also be packed witha hydrophobic support m, and once it is affixed to the outlet end d ofthe hydrophobic column, becomes an extension of the original hydrophobiccolumn upon which the sample is immobilized. As a result, the adaptermay be used only once and then must be discarded. Accordingly, there isa need for a sample column able to withstand the high pressures ofRP-HPLC that might be directly incorporated, without an adapter ormodification, into an HPLC receptacle so as to integrate the samplecolumn as the chromatography column of the RP-HPLC.

The above single-sample procedures associated with the current state ofthe art device are incapable of performing chemistries or sequential,uninterrupted treatment of multiple samples and results, therefore, inextremely tedious protocolsand prone to cross-contamination. Further, awell recognized problem associated with the incorporation of thehydrophobic column into the RP-HPLC is that the coupling between theinlet port of the column and the RP-HPLC line is such that residualliquids are trapped in the headspace between the end of the columnsupport material and the RP-HPLC coupling. It is well known in the artthat a zero-head space is required in order to assure the most accurateHPLC measurements. The existence of a non-zero head space introduceserrors into the HPLC analysis.

Chemical procedures and treatments performed on a sample in preparationfor analysis can become tedious, particularly where repetitivechemistries must be performed and time consuming where hundreds orthousands of samples are involved. Additionally, it also createssignificant opportunities for errors in measurement, and forcontamination of the sample or reagents. Further, if characterization ofthe sample requires several different chemistries to be performed thereis an increased chance of error as the technician must now identify,track, and monitor the progress of each of the required protocols.Although the present state of the art includes micro-controllerinterface with semi-automated apparatus, the technician must stilldetermine which chemical procedure or protocol is to be performed on anyparticular sample, and key that protocol selection into themicro-controller. If the technician executes the incorrect protocol, thesample is ruined at best, or, at worst the erroneous analytical datarecorded on that sample is included in the data being accumulated.

There is a need to provide an automated means for sequential,uninterrupted performance of chemistries on a plurality of samplescontained within or immobilized on a plurality of sample columns withminimal human intervention and reduced risk of performing incorrectprotocols.

SUMMARY OF THE INVENTION

This invention is directed to a cassette-type chemical treatment systemand method for enabling the performance of various chemistries withminimal human intervention, near-zero dead volume, and flow-throughprotocols pursuant to a predetermined instruction set encoded on thecassette.

The cassette comprises a plurality of sample columns ("mini-columns"),reagent wells, sample loading funnels, alignment means for the samplecolumns, and a machine readable instruction code set for determining achemical treatment protocol. All components are constructed from inertmaterials including but not limited to linear polyethylene,fluoro-copolymers, Teflon™, and the like.

The mini-columns contain a long inner chamber packed under pressure witha solid support, preferably silica, although other supports such aspolymer beads, resins, and cellulose may be used. Alternately, no solidsupport at all need be used when, for example, the sample is retained asa dry powder or beads, or if the sample is microencapsulated. In thepreferred embodiment, a silica support is employed and derivatized torender it hydrophilli or hydrophobic. For protein chemistries, thesilica support is derivatized with a lipophilic alkyl group thusrendering the support non-polar. Consequently, lipophilic proteinsdissolved or suspended in a more polar mobile phase will becomeimmobilized on the hydrophobic support. Unlike the mini-columns of theprior art, both terminal ends of the mini-columns of this invention aredesigned to accommodate the extremely high pressures (greater than 1200psi) needed to perform reverse-phase HPLC of the immobilized proteins orother hydrophobic moieties. This is accomplished by designing agenerally concave, preferably tapered opening at each end of the columnso that an interface means may be attached thereto with near-zero deadvolumn. The opening can be spheroidally concave, parabolic, conicallytapered, and the like so long as a high pressure seal can be effectedwith a compression fit interface means. Suitable compression fitinterface means include: a nozzle having a rounded tip for insertion andcompression fit against the walls of the generally concave, preferablytapered opening; a nozzle having a conical tip for insertion inside theinner diameter of the longitudinal chamber for compression fit againstthe junction of the inner wall of the longitudinal chamber at apex ofthe generally concave, tapered opening with the conical nozzle; and thelike to permit localized interface wit the inlet and outlet ports toenable near-zero dead volume communication. The term "localizedinterface" in the context of this invention refers to a near-zero deadvolume interface wherein the surface area of the connection does notextend beyond the generally concave, preferably tapered walls of themini-column opening, thus significantly reducing or minimizing thesurface area in which reagents, solvents, and detached samples mightcontact. This is contrasted to the prior art where introduction ofreagents and solvents is by dispensing the reagent or solvent into thesame sample funnel resulting in a significant risk ofcross-contamination. In the preferred embodiment, the distal end of arounded nozzle is inserted into the generally concave, preferablytapered opening, seated against the sides of the taper, and urged withsufficient force to provide a pressure tight seal so as to withstandcolumn pressures up to 2000 psi. This feature permits the column of thisinvention to be directly integrated as the support column in areverse-phase HPLC apparatus. The outlet end of the mini-column may alsocontain a generally concave, preferably tapered shoulder to mate with analignment assembly, described below.

As described above, each mini-column may be loaded with a hydrophobicsupport. The ends of each column are fitted with a porous frit to permitliquid flow and to retain the solid support within the column chamber.The frit may be made from any inert material such as sintered glass,fluorinated polymers, and other polymers such as sintered polypropylene.

A novel feature of the cassette of this invention is that it includes aplurality of loading funnels to provide a funnel assembly. The funnelsmay be arranged in whatever configuration is deemed expedient in view ofthe ability of the treatment station to address each mini-column orreagent well opening with a nozzle. For example, if the funnel assemblyis arranged as a rectangular or square array, the treatment station mustprovide for nozzle arrays corresponding to the X-Y location of eachinlet and outlet port for each column and well. The inlet end or loadingport of each mini-column is press-fit disposed in a connection sleeveextending from the throat of a corresponding sample loading funnel ofthe funnel assembly. The taper of the inlet port of the mini-column iscoplanar with the taper of the conical bottom of the funnel so thatthere is a virtually seamless transition from the funnel to the inletport of the mini column. This will ensure that no dead volume exits thatmight lead to possible cross-contamination. Alternately the mini-columnmay be integrally cast as an extension of the loading funnel. In thefirst embodiment, once the sample on the column has been treated, thecolumn may be removed for insertion into the high pressure line of anexternal HPLC analyzer such that the mini-column is now the supportcolumn for the HPLC. This is contrasted to the column of the prior artwherein the mini-column is merely a pre-column, requires an adapter, anddoes not supplant the standard HPLC column. Alternately, an HPLCanalysis capability may be built into the system of this invention. Inthis case, the mini-columns need not be removed from the cassette andmay be remain in place after treatment of the sample. The same nozzleinterface used to address each column now acts as part of the highpressure line for an integrated HPLC.

In order to ensure the mini-columns remain press-fit connected to thefunnel assembly and to ensure that each mini-column is aligned with theaxis of symmetry of its corresponding funnel, an alignment means isoptionally provided. The alignment assembly of the cassette of thisinvention ensures that the longitudinal axis of all mounted mini-columnsare coincident with the longitudinal axis of its corresponding loadingfunnel. Proper alignment is essential, as will be explained below, toensure that both the inlet port and outlet port of each mini-column isproperly aligned to receive the distal ends of dispensing and expendingnozzles although self-centering is achieved by virtue of the generallyconcave, preferably tapered openings. Where the mini-column is notintegral to the funnel assembly, an alignment assembly may be provided.Once each mini-column has been inserted into the funnel assembly, thealignment assembly is positioned over the outlet ports of themini-column. The alignment assembly is aligned with and removeablyattached to the funnel assembly as a spatial reference. Precisely spacedthrough-holes having a tapered inner bore are positioned on thealignment assembly to mate with the tapered shoulder of outlet port ofeach mini-column. Each mini-column is now secured on each end, thusholding each mini-column in proper alignment. Alternately, an extendedsleeve maybe cast as part of the funnel assembly so that insertion ofthe mini-column into the extended sleeve will result in a stable orrobust alignment as well as providing sufficient press fit of themini-column to prevent the mini-column from falling out.

Where the mini-column is an integral part of the funnel assembly,bracing sufficient to ensure alignment of the mini-columns may cast aspart of the integrated funnel assembly. For example, cross-bracingextending from the external wall of the integral mini column to thefunnel assembly will provide a stable or robust alignment of themini-columns.

A novel feature of this invention is the optional integration of aplurality of reagent wells into the funnel assembly. The reagent wellsare open on each end and are supplied with frits, as in themini-columns, to prevent material contained in the wells from fallingout. The reagent well may be packed with a suitable support forimmobilizing the specific reagent contained therein. Alternately, thereagent may be in powdered form either as a soluble solid or as alyophilized solid. The solid reagent may be provided as amicroencapsulated reagent (this would permit use of liquid reagentswithout having to provide a solid support), as beads of a predeterminedsize to permit solvent flow through and/or controlled solubility rates,or, alternately, the solid reagent may be painted on the walls of thecolumn chamber, thus permitting the free flow of solvents.

Alternately, the reagent wells may be used as an alternative way tointroduce samples for chemical treatment. By packing the wells with asuitable solid support, sample may be immobilzed on the support. Thiswould effectively double the number of samples available on the cassettefor treatment. Sample in other forms, such as adsorbed, lyophilized,powdered, microencapsulated, or free liquid, may be placed in the samplecolumns, scanning the reagent wells with a scanning means will identifypopulated wells whose contents might participate in the predeterminedchemical protocols.

As in the mini-column is, the ends of the inside chamber of the reagentwells are generally concave, and preferably tapered to provide a flaredopening to permit the pressure tight seating of the distal ends of adispensing and expensing interface means. These interface means includethe same means discussed supra; e.g., nozzles having rounded or conicalshaped tips. Once the fully assembled and loaded cassette assembly isinserted into the treatment system of this invention, each column andwell opening is addressed by a nozzle. The tip of the nozzle is of ageometry designed to fit within the generally concave, preferablytapered openings of each mini-column and reagent well, and provide atight seal thus achieving near-zero dead volume resulting in minimizingthe risk of cross-contamination. In contrast, the prior art systems,which have no nozzle interface, or other direct flow-throughcommunication interface, has a significant amount of dead volume. Anovel feature of this invention is that the benefit of a funnel isattained for sample loading, however, near-zero dead volume is attainedas well by the use of the nozzle interface. In accordance with thepreselected chemical treatment protocol identified for a particularcassette, the nozzles permit the free flow introduction of a solvent,treatment solution, or a sample-containing solution to the column orwell with which it is in communication (dispensing) or for removal of asolvent, spent treatment solution, or sample containing solution from acolumn well with which it is communication (expensing).

Another novel feature of the cassette of this invention is that eachcassette has a machine readable code disposed on the cassette that isread by an appropriate device in the treatment station to automaticallyindicate to the treatment station in which the cassette is loaded theexact chemical protocols required for the samples in the cassette. Thecode will optionally indicate whether or not reagent is present in thereagent wells, whether the reagent wells contain additional samplesrather than reagent, and which column and well addresses are to treated(in the event that not all column and well addresses are populated).Alternately, a scanning means can scan each column and/or reagent wellto identify those column and reagent addresses that have material; i.e.,solid support, reagent, solvent. Accordingly, empty columns and wellsare not addressed and only those populated columns and wells identifiedby the scanner means participate in the identified chemistries. Scanningmeans suitable for identifying populated mini-columns and reagent-wellsinclude either a light array having a single source or a plurality ofsources and complementary light source detector array, or a mechanicalprobe inserted into the mini-column and reagent well openings to sensethe presence of an obstruction such as a frit. In the preferredembodiment, the light source array is positioned over the mini-columnand reagent well ports while the detector is positioned to detectwhether the light is transmitted through the mini-column or reagentwell. Those "empty" mini-columns and reagent wells where the light istransmitted through the column or well do not participate in theidentified predetermined chemical protocols.

The machine readable code may be in the form of a bar code, a magneticstrip, an embedded diode, or a semiconductor memory chip. The deviceused to read the code will necessarily depend on the format and mediumof the code and may include a bar code reader, a magnetic strip reader,a radio transponder, or a data bus socket. The foregoing means forencoding as machine readable code the chemical protocol information andthe scanning devices for reading the machine readable code are presentedby way of example and not by way of limitation as any means whetheroptical, magnetic, electrical impulse, and the like may be employed toprovide to the treatment station an indication of the desired chemicalprotocols. A further novel feature of the cassette of this invention isthat once the cassette has been loaded into the treatment station ofthis invention the machine readable code is modified to indicate thatthe desired chemical treatment protocol has been performed. Suchmodification may include modifying the code so that it becomesunreadable by the scanning device, thus preventing execution of anytreatment protocol. For example, a bar code may be disposed in a barcode holder that is slideably inserted in a receiving groove in thecassette. Once the bar code is read, the bar code holder may berepositioned in the receiving groove so that part of the bar code ispositioned in a pocket in the cassette, thus obscuring at least aportion of the bar code, rendering the bar code unreadable. This willserve to prevent the cassette from being inadvertently processed asecond time. As a further example, in the case of the semiconductormemory, a CMOS or static RAM may be used to contain the requiredprotocol instructions. Once the memory is read, the coded instructionset may be modified to indicate that the cassette has undergone theprescribed chemical protocols. An advantage of using a random accessmemory means is that the modification of the machine readable code mayoptionally include writing information to the memory to provideinformation as to date and time the protocols were executed, the name ofthe technician operating the system, any deviations to the protocol,addresses of the columns and wells, and parametric information such asreagent volumes, operating temperatures and pressures, and the like.Optionally this information may be later downloaded to a permanentinformation storage location.

The system of this invention includes the cassette assembly, a sampleloading station, and a treatment station. By way of operation, thecassette assembly is loaded into the sample loading station and alignedin preparation of receiving the sample. An annular gasket is loweredonto the top rim of the sample funnel being loaded. The pressureresulting from mounting the loading ket is such that the mini-columnassociated with the instant sample funnel is firmly pressed against thefunnel opening to ensure a water tight fit between the narrow end of themini-column and the sample funnel connection sleeve up to about 40 psi.The outlet end of the mini-column is position over a drain tube. Thesample solution is then pipetted into the first well. After the samplesolution has been introduced, a pressure cap having a centrally disposedplunger is lowered over the gasket so that the plunger extends into thesample funnel. An annular shoulder on the plunger seats against thegasket to create an air tight seal up to about 40 psi. A port in theplunger permits an inert gas to pressurize the head space in the samplefunnel thus forcing the sample solution into and through themini-column. Alternately, the sample solution may be forced into andthrough the mini-column by either providing a vacuum draw at the outletend of the mini-column to suction the sample solution through, or theplunger on the pressure cap may directly push the sample solutionthrough the mini-column; i.e., hydraulic pressure. As the samplesolution passes through the mini-column, any lipophilic moieties areimmobilized on the hydrophobic packing material. Similarly, if thepacking material were hydrophilic, any lipophobic moieties would beimmobilzed in that case. Superfluous sample solution is expired throughthe outlet port into a drain line. The funnel is depressurized, and theplunger and gasket removed. This process is repeated for all samplefunnel/mini-column addresses in the array.

Alternately a multi station sample loading device may be used wherebyall sample funnel/mini-column addresses are loaded simultaneously. Thisembodiment, as well as the single station loading station embodiment,may optionally provide an sample dispensing port in the plunger(s) toautomatically dispense the sample solution into the sample funnelwell(s) after the plunger is seated. A further option includes a meansfor reading the machine readable code so that a microcontrollerinterface might load the cassette pursuant to a predetermined samplingprotocol.

OBJECTS AND ADVANTAGES

It is an object of this invention to provide a cassette for a chemicaltreatment system having a plurality of funnels for loading samplesolutions, a plurality of sample retaining means for holding a pluralityof addressable, preselected samples and preselected reagents, to permitpreselected chemistries on the preselected samples in a sequentialinterrupted fashion, the cassette having a machine readable codeintegrated thereon to automatically identify to the chemical treatmentsystem upon insertion of the cassette into a chemical treatment stationthe chemistries to be performed on the preselected samples using thepreselected reagents, the cassette having very near-zero dead volumeflow-through connection with a chemical treatment station.

It is another object of this invention to provide a sample loadingstation not requiring pre-isolation of the sample in a reaction chamberand that will enable rapid loading of the chemical treatment cassette ofthis invention.

It is another object of this invention to provide an improved samplecolumn for sample immobilization or containment and for insertion intothe cassette of this invention to permit flow-through chemistries withnear-zero dead volume and to permit access to and use of the improvedsample column as an HPLC column without the need for a high-pressureadapter.

It is another object of this invention to provide a sample treatmentstation for receiving the chemical treatment cassette of this invention,for reading the machine readable code on the cassette, foruninterrupted, sequential accessing all pre-selected samples andpre-selected reagents, and for executing the chemistries as identifiedby the machine readable code.

It is another object of this invention to provide a chemical treatmentsystem having a sample loading station, a chemical treatment cassette,and a chemical treatment station for performing automatic,near-simultaneous, flow-through chemistries on a plurality ofpre-selected samples using a plurality of pre-selected reagents, thechemistries being identified by machine readable code to amicro-controller, and the chemistries being executed by themicro-controller working in logical and electrical cooperation with thechemical treatment station.

It is another object of this invention to provide a method for nearsimultaneous performance of chemistries on a plurality of preselectedsamples, the samples being retained in a chemical treatment cassette,and the chemistries desired retained on machine readable code forautomatic execution via micro-processor control pursuant to instructionscontained in the machine readable code.

Still other objects will be evident from the specification claims anddrawings of this application.

BRIEF DESCRIPTION OF DRAWINGS

The invention is illustrated by reference to the drawings in which:

FIG. 1 is a cross-sectional diagram of a single-sample chemicaltreatment cartridge or reactor of the background art;

FIG. 2 is a cross-sectional diagram of the sample column of thebackground art as connected to an HPLC high-pressure adapter;

FIGS. 3a-e are various perspective views of the chemical treatmentcassette of the chemical treatment system of this invention;

FIG. 3f is a cross section view of the multiple sample, chemicaltreatment cassette cassette assembly of this invention;

FIG. 4 is a cross-sectional diagram of the sample mini-column of thisinvention;

FIGS. 5a,b are cross-sectional views of the chemical treatment cassetteof the chemical treatment system of this invention showing therelationship of the sample columns and nozzles with respect to eachother and to other elements of the chemical treatment cassette;

FIG. 6 is a cross-section view of an equally preferred embodiment of thechemical treatment cassette of the chemical treatment system of thisinvention;

FIG. 7 is a cross-section view showing how the nozzle is seated in thetapered opening embodiment of the mini-columns and reagent wells of thechemical treatment cassette of this invention;

FIG. 8 is a cross-section view of the chemical treatment cassette asloaded in the sample loading station;

FIG. 9 is a perspective view of the chemical treatment cassette and theupper and lower nozzle arrays of the chemical treatment station andtheir relative positions spatial positions prior to loading the nozzlesinto the sample column and reagent well generally concave, preferablytapered openings;

FIG. 10 is a schematic overview diagram of the hydraulic layout of thechemical treatment station of this invention;

FIG. 11 is a cross-section diagram of the mini-column of this inventionbeing in an HPLC in-line adaptor; and

FIG. 12 is a flow diagram of the method of using the chemical treatmentsystem of this invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The following detailed description illustrates the invention by way ofexample, not by way of limitation of the principles of the invention.This description will clearly enable one skilled in the art to make anduse the invention, and describes several embodiments, adaptations,variations, alternatives and uses of the invention, including what wepresently believe is the best mode of carrying out the invention.

The three principle elements of the chemical treatment system of thisinvention include a sample loading station, a sample treatment station,and a chemical treatment cassette. Both the sample loading station andthe chemical treatment station elements are configured in accordancewith the geometry and configuration of the chemical treatment cassette(hereafter "cassette"). The preferred embodiment of the cassette iseasily seen in the exploded view as shown in FIG. 3a. The principleelements of the cassette 10 include a sample funnel assembly 11, aplurality of mini-columns 13, a mini-column alignment assembly 15, and areceptacle for removeably retaining a medium containing a machinereadable code 17.

The sample funnel assembly 11 contains a plurality of funnels 19integral to the assembly and arranged in a even, regular funnel array.FIG. 3a shows the sample funnel assembly as a linear array 21. FIG. 3bshows the mini-columns 13 inserted into the funnel assembly 11. Thelocation of each mini-column may be stamped onto the alignment assembly15 to facilitate sample loading and for tracking purposes. FIG. 3c is amirror-image perspective view of FIG. 3b and clearly shows the pluralityof through-holes 23 in the alignment assembly by which the flared,generally concave, preferably tapered, free end of the mini-column maybe aligned for later connection and interface to a chemical treatmentstation. The alignment holes 23 are tapered so as to receive the taperedflange of the free end of the mini-columns and to self align the columnsas the alignment assembly 15 is snapped into place.

FIG. 3c shows the inlet ports 29 of reagent wells 31, shown in phantom,that are integrated into the funnel assembly 11. As discussed above, thereagent wells may be used as a source of adsorbed, powdered, freezedried, microencapsulated, and liquid reagents, solvents, salts, buffers,or other chemicals participating actively or passively in the chemistryperformed on the samples in the mini-columns.

Also shown in FIG. 3c is the receiving slot 17 for the machine readablecode means. In the preferred embodiment of the cassette of thisinvention the machine readable code means is a bar code. FIG. 3d showshow a bar code 25 is inserted into the receiving slot 17. FIG. 3e showsthe bar code 25 slideably received in the receiving slot. As shown inphantom, the bar code 25 has been displaced to the extreme bottom end 27of the receiving slot 17 rendering the bar code unreadable. A mechanismis provided in the treatment station of this invention to displace thebar code to the extreme bottom end of the receiving slot once thecassette has been addressed by the interface nozzle arrays contained inthe treatment station by which the flow-through chemistry is effected,thus preventing the cassette from inadvertantly being used again.

A cross-section view of the chemical treatment cassette assembly isshown in FIG. 3f which clearly shows the inter-relationships between thevarious elements of the cassette assembly. The sample mini-column 13 hasa first narrow end 33 which is press-fit into a mini-column connectionsleeve 35 formed by extending the throat of the sample funnel 19. A stopshoulder 37 disposed annularly on the funnel end of the inside wall ofthe connection sleeve 35 provides a stop barrier to prevent themini-column 13 from extending into the cavity of the funnel 19 andprovides a sealing surface between the inlet port of the column and thethroat of the funnel to prevent sample solution from leaking duringsample loading. Further, the stop barrier extends over the edge of themini-column narrow end 33 by an amount sufficient to provide a smoothtransition from the sloped inside wall 39 of the funnel to the generallyconcave, preferably tapered wall 41 of the narrow end of themini-column. This smooth transition from funnel to mini-column reducesthe risk of sample or wash solvent residues forming on what wouldotherwise be a surface irregularity. Such residues may lead to errorsand significantly affect the results of the chemistries involved.

FIG. 3f also clearly shows the structure of the reagent wells 31. Thewells have an inlet port 29, and an outlet port 43. A center chamber 44runs longitudinally through the reagenet well and connects the inletport 29 with the outlet port 43 within which chamber may be disposed anyreagents, solvents, buffers salts, enzymes, and the like, in either anadsorbed, powdered, lyophilized, microencapsulated, or liquid state,useful in the chemistries associated with the sample that is retained bythe sample mini-column 13. Inert porous frits 45a and 45b are press fitinto the ends of the central chamber to prevent loss of the materialscontained in the reagent well central chamber.

FIG. 3f also clearly shows how the flange 49 of the flanged end 47sample mini-column 13 is seated in the through hole 23 of the alignmentassembly 15. The outside annular edge 51 of the flange 49 is tapered sothat it mates with the taper of the inside wall of the through hole 23.Once the alignment assembly 15 is snapped into place with the funnelassembly 11, the mini-column is held in an aligned position stableenough to move and manipulate the cassette assembly 10 without looseningthe mini-columns 13 from their connection sleeves 35. The mini-column 13of this invention is shown more clearly in FIG. 4. It is comprised of anarrow end 33, a flanged end 47 having an annular flange 49 outwardlyextending from the flanged end 47. The outside outside annular edge 51of the flange 49 is tapered inwardly so as to be accomodated by andself-centering in an alignment hole 23 of the alignment assembly 15, thealignment hole also having a complementary tapered bore for receivingthe outside tapered mini-column flanged end 47. A logitudinal chamber 59connects a narrow end inlet port 53 with a flanged end outlet port 55.Both the inlet port 53 and the outlet port 55 have a bores 41 and 57 tofacilitate leak-proof, high pressure seal with a nozzle interface of thechemical treatment station of this invention or external analyteanalyzer. The chamber is typically packed with a solid support material61 such as silica that has been derivatized with a lipophyllic polymer(e.g., a C18 compound) thus rendering the support hydrophobic. Othersupport materials may be used including polymer or resin beads,cellulose, and the like. Further, depending on the sample to beimmobilized, the support may be made hydrophillic. The support materialis retained in the column by porous frits 63 and 65. These frits may bemade of any inert porous material including sintered polyethylene,polypropylene, fluoropolymers, glass, and the like.

FIG. 5a is an exploded, cross-section view of the chemical treatmentcassette assembly 10 showing how the nozzles of the chemical treatmentstation interface addresses the cassette. Each address of the cassetteincludes a reagent well 31 with an inlet port 29 and outlet port 43, anda sample mini-column 13, also having an inlet port 53 and an outlet port55. As is clearly shown in FIG. 5a, a mini-column inlet nozzle 67interfaces with the mini column inlet port 53 by seating the nozzle tip68 against the generally concave, preferably tapered bore 41 of themini-column inlet port 53. The nozzle 67 contains a through-bore 69disposed on the longitudinal axis of the nozzle, the through-bore havinga rounded, polished distal end 68 in communication with the inlet port53 of the mini-column through which solutions and solvents are eitherintroduced to or removed from the mini-column. Similarly, themini-column oulet nozzle 73 interfaces with the mini-column outlet port55 by seating the rounded, polished nozzle tip 74 against the generallyconcave, preferably tapered bore 57 of the mini-column outlet port. Thenozzle 73 contains a through-bore 75 disposed on the longitudinal axisof the nozzle, the through-bore terminating at the rounded, polisheddistal end 74 to provide communication with the outlet port 55 of themini-column through which solutions and solvents are either introducedto or removed from the mini-column.

Similarly, the reagent well inlet port 29 interfaces with a reagent wellinlet nozzle 78 having the same structure as the mini-column inlet portnozzle 67, and the reagent well outlet port 43 interfaces with a reagentwell outlet nozzle 79 having the same structure as the mini-column inletport outlet nozzle 73, to enable hydraulic and pneumatic communicationof the reagent well with the chemical treatment station.

Note that other interface means may be used including slip fittings,threaded fittings, gasketted butt-joint fittings, and the like, however,the preferred embodiment uses the nozzles as described above. FIG. 5bshows the nozzle tips inserted into the generally concave, preferablytapered bored openings of the mini-column and reagent well inlet andoutlet ports. FIG. 7 is a detailed cross-section view of the nozzle tip74 of the mini-column outlet port nozzle 73 seated in the mini-columnoutlet port 55, and typifies the nozzle/port interface of thisinvention. The rounded, polished end 74 of the nozzle 73 is urged orpressed against the generally concave, preferably tapered wall 57 of theoutlet opening 55 with a pressure sufficient to provide a leak-proof,seal between the tapered opening and the nozzle tip. The seal betweenthe reagent well and the reagent well nozzles must withstand up to 40-50psi, whereas the mini-column seals must withstand pressures in excess of1500 psi. The mini-column/nozzle interface must withstand these higherpressures to provide in-line HPLC capability.

The adavantage of the column/nozzle interface over the background art iseasily discerned. A significant amount of dead volume is introduced bythe system of the background art since the walls of their sample funnelprovide an extensive surface area on which residues and othercontaminants might collect. These residues pose a significantcross-contamination threat where multi-step chemistries are performed onless than milligram quantities of sample. By eliminating the samplefunnel as a means for dispensing reagents and solvents to themini-column (as in the background art) the interface of this inventionachieves a near-zero dead volume since there are no surfaces on whichresidues of prior solutions might collect, while still obtaining thebenefit of the funnel for sample loading.

FIG. 6 is a cross-section, exploded view of an equally preferredchemical treatment cassette assembly wherein the mini-column 14 at eachaddress is integral to the sample funnel assembly. This embodiment ispreferred when, for example, the chemical treatment station is capableof performing on-board, in-line analysis of the reacted sample, thusvitiating the need to remove the sample mini-column from the chemicaltreatment cassette (in order to perform off-line analysis of the reactedsample immobilized on the column). This embodiment requires no alignmentassembly as each integral column is prealigned and permanently affixedin place. Further, this embodiment permits the mini-column inlet andoutlet nozzles to act as the high-pressure interface connection with theon-board analyzer, typically HPLC, thus eliminating the need to have aseparate analyzer dock with a separate interface assembly in thechemical treatment station.

The loading station of this invention comprises an X-Y alignment means,a gasket loading means and a plunger or pressure cap means. FIG. 8 is across-section view of the chemical treatment cassette of this inventionin the sample loading station. The cassette is positioned so that thegasket and pressure cap will precisely engage the sample funnel. FIG. 8shows the cassette positioned via an alignment means showndiagramatically as an alignment pin 81 extending upwards from theloading platform 83. A plurality of alignment pins may be positioned onthe platform to permit precise positioning of the cassette assembly.Alternately, the cassette may be locked onto a slideable platform havingpredetermined stops to permit the precise positioning of each samplewell 19 for single sample loading. Alternately, an alignment stop havinga shape complementary conforming to the external shape of the funnelassembly may be used to position and index the individual funnels duringsample loading. Once positioned, an annular gasket 87 is positioned overthe sample well and a seal is made by exerting a downward pressure onthe gasket to provide a pressure seal. The pressure seal also serves tourge the mini-column outlet port against a drain interface 85 thusproviding a seal between the outlet port and the drain interface, and tourge the narrow end 33 of the mini-column 13 against the annularshoulder 37 in the connection sleeve 35, thus providing a pressure sealbetween the sample funnel 19 and the mini-column. Next, the samplesolution 16 is introduced into the sample funnel 19. The pressure cap 89is then lowered onto the gasket and pressure applied to provide a sealbetween the pressure cap and the gasket. A centrally disposed plunger 90on the pressure cap extends into the funnel 19. Inert gas is thenintroduce into the head space 18 between the sample solution and theplunger 90 via a gas entry port 91 disposed in the pressure cap, thusforcing the sample solution 16 through the mini-column 13 with theexcess solution passing through the mini-column and drained away throughthe waste tube 86 connected to the drain interface 85. Once the samplesolution has passed through the column, the inert gas pressure isreleased, and the pressure cap and gasket are removed. The cassette isthen repositioned for sample loading of the funnel at the next address,or optionally, either a second sample solution or a wash solvent may beloaded. This process is repeated until all the mini-columns at thedesired addresses have been loaded. It should be noted that the loaderof this invention does not require that the sample funnel or thecassette be inserted in a special reaction chamber or holder in order toload the mini-column as is done in the background art. Further, thenovel aspects of the loader of this invention may be extended to amultiple sample loading station whereby more than one address may beloaded with an sample at a time. Also, an optional sample introductionport 93 may be provided in the pressure cap so that the gasket 87, whichserves to pressure seal the mini-column with the funnel prior tointroduction of the sample, may be eliminated since the pressure capwill now provide the force required to seal the mini-column against thefunnel and the sample is not introduced into the funnel until after thepressure cap is in place.

After the chemical treatment cassette has been loaded it is insertedinto the chemical treatment station (CTS) of this invention. The CTSpositions the cassette so that the cassette/CTS interface can beestablished. Positioning of the cassette is performed automatically bymeans well known in the art. FIG. 9 is a diagram showing the cassette 10position between the nozzle interface arrays 95 and 96. After insertioninto the CTS, the bar code 25 is read by a bar-code reader 99. Theinstructions contained in the bar code are sent to a micro-controllerand the appropriate algorithm is accessed and loaded for execution ofthe process steps and chemistries indicated by the bar codeinstructions. Once the cassette is transferred into position, interfacenozzle arrays 95 and 96 are brought together and into contact with thecassette so that the tips of the nozzles 67, 78, 73, and 79 are broughtinto pressure contact with their respective mini-column and reagent wellports. As the nozzle arrays are brought together, the bar code 25 isdisabled by action of a tab or pin 96 on the slideable bar code forcingit to a position in the bar code receiving slot 17 where the bar code isat least partially obscured. Once the nozzle/cassette interfaces havebeen established the materials in the reagent wells and the immobilizedsample in the minicolumns are now accessible to the CTS so that thepredetermined chemistries can be performed.

The preferred embodiment of the CTS of this invention is shownschematically in FIG. 10. The loaded chemical treatment cassette isdepicted as being addressed by the nozzle array interfaces. Themini-columns are addressed by the mini-column inlet port nozzles 67 andoutlet port nozzles 73, and the reagent ports are addressed by inletnozzles 78 and outlet nozzles 79. Each reagent well may be selectedindividually for processing by adjustment of rotary valves 100, 101,103, and 104, and each mini-column may be selected individually by theappropriate adjustment of rotary valves 105, 106, 107, and 108. Notethat rotary valve inlet and out let pairs; i.e., 100 and 103, 101 and104, 105 and 107, and 106 and 108, are synchronized so that when theinlet port of a reagent well or a mini-column is selected, the rotaryvalve for the outlet port may only be set to that reagent well ormini-column.

Solvents, reagents, buffers, and other solutions are supplied from up tosix reagent bottles 111a-f, with the desired reagent being selected byappropriate adjustment of the solvent valve blocks 109 and 110, and byappropriate selection of pressure switch from the pressurized valveblocks 122 and 123. These reagents are in addition to the reagentssupplied in the reagent wells of the cassette. All solvents must passthrough valve V4 102. Default position for all solvent and reagentrotary valves is set to direct the reagents to waste 112.

Reagents 111a-f may be directed to the mini-columns by closing valve V102. This will direct the selected reagent to the mini-column inletselected by the rotary valves 105 and 106 with any waste reagent passingthrough valve 118, valve 119 and into the waste bottle 112.

Solvents may be directed to the reagent wells for rehydration orsolvation of adsorbed, powdered or lyophillized reagents in the reagentwells by switching rotary valve V4 102. Solvent is now directed toreagent cell inlet rotary valves 100 and 101. Note that postion 6 of thereagent well and mini-column rotary valves is a pass-through position.Thus if rotary valve 100 is set at position 6, the solvent will passthrough to the reagent selected by rotary valve 101. If desired, thesolvent may be passed-through and stored in the mixer-diluter 114 byclosing valve V8 115 and V4 102. This permits mixing various reagentsand solvents with one another, including solvated dry reagents from thereagent wells of the cassette, prior to moving the mixture through line124, valve 117 and valve 116, and to the mini-column rotary valves 105and 106 for reaction with the immobilized sample.

The CTS of this invention provide three areas where solvent, analyte orsample solutions may be stored. These areas include the mixer diluterwhich as described above, permits solvents and reagents to be mixedprior to reaction with the sample or analyte. Reagents, sample andanalyte may also be accumulated (i.e., temporarily stored) in the heatercoil 126 and in the shaking coil 125. It may be desireable to remove tiesample or analyte from the support packing in the mini-column for anumber of reasons. For example, where the hydrophobic support results ina conformational deformation of a protein or peptide side chain orotherwise affects the reactivity of the peptide or protein side chains,it might be desireable to perform the desired reaction external to thecolumn. In this event, a suitable lipophobic solvent is used to detachthe protion from the support. Valves V10 119, and valve 117, and 116 areclosed resulting in the sample or analyte, now in solution, to be forcedout of the inlet port of the affected mini-column, through valve 117,heater coil 126, valve 116 and into the mixer/diluter 114 wherein thedesired reaction chemistry may be performed with reagents alreadypresent, or introduced later. As another example, detaching the samplemay be desireable in the event the characteristics of the supportmaterial must be changes; e.g., changing it from a hydrophobic supportto a hydrophillic support in the middle of the chemistry beingautomatically executed by the CTS.

The CTS of this invention is able to mix solvents, reactants andbuffers; to detach and shuttle the sample back and forth (i.e., pump-upand pump-down; to perform complex chemistries either on or off-column;and to solvate adsorbed, powdered or lyophillized reagents, allperformed automatically, pursuant to instructions indicated to amicro-controller by a bar code, without having to remove the cassetefrom the CTS, without having to detach or reattach the CTS/cassetteinterface, or otherwise require human intervention. Further, the samechemistries may be performed on all sample mini-column addresses, or aseparate completely independent set of protocols may be defined for eachaddress, or for each block of addresses.

Further, an optional analyte analysis capability may be included in theCTS of this invention. Any of the mini-columns may be converted to anin-line HPLC column, by swithing rotary valve 117 to receive solventfrom an HPLC pump and by switching rotary valve 118 to direct the eluantto a detector. It is clear that the CTS is capable of performing avariety of analysis other than HPLC without having to remove thecassette from the CTS. However, if on-board, in-line analysis is notavailable, the mini-column may be removed from the cassette and directlyinserted into an in-line, high-pressure adapter as shown in FIG. 11. Theadapter 130 receives the mini-column 13 containing the analyte. Nozzles131 and 133 are seated against the generally concave, preferably taperedinlet port 53 and outlet port 55 to form a high pressure seal. Themini-column of this invention does not require that a separatecolumn/adapter be provided in order to accomodate in-line HPLC.

The process of this invention as described supra beginning with theloading of the cassette, followed by execution by the CTS of theselected chemistries as predetermined by the instructions contained on abar code affixed to the cassette, and concluding with either in-lineonboard analysis, or an improved external analysis is summarized in FIG.12.

It should be understood that various modifications within the scope ofthis invention can be made by one of ordinary skill in the art withoutdeparting from the spirit thereof. We therefore wish our invention to bedefined by the scope of the appended claims as broadly as the prior artwill permit, and in view of the specification if need be.

I claim:
 1. A unitary, one-piece mini-column for retaining a chemicalsample for chemical treatment, said mini-column comprising a centrallydisposed chamber extending longitudinally through a cylindrical column,said chamber terminating into a first open end, said first open endhaving a flared, tapered inlet port, said first open end enabling slipfit, flow-through, physical connection with a connection sleeve, saidconnection sleeve extending from a through-hole in the tapered, conicalend of a sampling funnel, said connection resulting in smooth,continuous transition of a taper from said conical end of said funnel tothe taper of said inlet port, said chamber terminating into a secondopen end, said second open end having a flared, tapered outlet port,said outlet port being in flow-through communication with said inletport, said tapered inlet port and outlet port each providing localized,leakproof communication with a compression fit interface means.
 2. Amini-column as in claim 1 further comprising a solid support materialdisposed in the central chamber, said solid support material beingtreated to immobilize said chemical sample, said solid support materialbeing retained in said mini-column by porous frits disposed in saidcentral chamber.
 3. A one-piece column for retaining a chemical sample,said column comprising a centrally disposed chamber extendinglongitudinally through a cylindrical column, said chamber terminatinginto a first open end, said first open end having an inlet port having ataper, said first open end enabling physical connection with aconnection sleeve extending from a through-hole in the end of a samplingfunnel, said connection resulting in transition from said funnel to thetaper of said inlet port, said chamber terminating into a second openend, said second open end having an outlet port, said outlet port beingin communication with said inlet port, wherein said one-piece columndefines at least a single bevel surface.
 4. A one-piece column forretaining a chemical sample, said column comprising a centrally disposedchamber extending longitudinally through a cylindrical column, saidchamber terminating into a first open end, said first open end having aninlet port having a taper, said first open end enabling physicalconnection with a connection sleeve extending from a through-hole in theend of a sampling funnel, said connection resulting in transition fromsaid funnel to the taper of said inlet port, said chamber terminatinginto a second open end, said second open end having an outlet port, saidoutlet port being in communication with said inlet port, wherein saidinlet port defines a bevel sealing surface.
 5. An arrangement forholding chemical samples, including a funnel and a one-piece column forretaining a chemical sample, said column comprising a centrally disposedchamber having a first open end, said first open end having an inletport having a taper, said first open end enabling physical connectionwith a connection sleeve extending from a through-hole in said funnel,said connection resulting in transition from said funnel to the taper ofsaid inlet port, said chamber terminating in a second open end, saidsecond open end having an outlet port, said outlet port being incommunication with said inlet port, wherein said inlet port defines abevel sealing surface.
 6. An arrangement for holding chemical samples,including a funnel and a one-piece column for retaining a chemicalsample, said column comprising a centrally disposed chamber having afirst open end, said first open end having an inlet port having a taper,said first open end enabling physical connection with a connectionsleeve extending from a through-hole in said funnel, said connectionresulting in transition from said funnel to the taper of said inletport, said chamber terminating in a second open end, said second openend having an outlet port, said outlet port being in communication withsaid inlet port, further comprising first and second bevel surfaces. 7.The arrangement according to claim 6 wherein said first bevel surface isadapted for sealing.
 8. The arrangement according to claim 6 whereinsaid second bevel surface is adapted for sealing.